The objective of this proposal is to extend our studies on several brain endopeptidases and to define their role in neuropeptide metabolism. One of these enzymes, prolyl endopeptidase, is highly active in brain. The enzyme is likely to be involved in neuropeptide metabolism since it cleaves bonds after proline residues, which are present in many neuropeptides and their precursors. We will use kinetic studies to determine the binding affinity and catalytic efficiency of the enzyme toward a variety of bioactive peptides in order to identify those peptides having the highest affinity toward the enzyme. N-benzyloxycarbonyl-prolyl-prolinal, a potent active site-directed inhibitor (Ki = 14 nM), recently synthesized in our laboratory, will be used to probe in vivo the role of prolyl endopeptidase in neuropeptide degradation. Since deamidation of thyrotropin releasing hormone (TRH) by prolyl endopeptidase terminates its function, we will study the effect of the inhibitor on endogenous brain and pituitary TRH levels, and on serum thyrotropin levels, expected to be influenced by inhibition of TRH degradation. Extension of similar studies to other neuropeptides is also planned. The concept that enzymatic degradation of neuropeptides may control their function will be tested in a pituitary GH3 cell line culture. These cells secrete prolactin after stimulation by TRH. The effect of the inhibitor on prolactin secretion by these cells will therefore be determined. Since degradation of TRH can also proceed by removal of the N-terminal pyroglutamyl residue catalyzed by pyrrolidone carboxylate peptidase, we will synthesize 5-oxoprolinal, a potent inhibitor of this enzyme, and study its effect on TRH degradation and function in a manner similar to that described for N-benzyloxycarbonyl-prolyl-prolinal. Studies will be continued on the multicatalytic protease complex discovered in our laboratory. Special attention will be directed toward studies on the component of the complex that is dramatically activated by low concentrations of sodium dodecyl sulfate and several natural fatty acids. The specificity of this component toward synthetic and natural peptides will be studied in detail. Antisera obtained in our laboratory against prolyl endopeptidase and the multicatalytic protease complex will be used in a collaborative study to localize these enzymes in the CNS by immunohistochemical methods. Neuropeptide metabolizing enzymes represent a potential target for pharmacological intervention. Specific inhibitors of these enzymes may profoundly affect the physiological processes mediated by neuropeptides.